As indicated above, the MN gene and protein are known by a number of alternative names, which names are used herein interchangeably. The MN protein was found to bind zinc and have carbonic anhydrase (CA) activity and is now considered to be the ninth carbonic anhydrase isoenzyme—MN/CA IX or CA IX [4]. According to the carbonic anhydrase nomenclature, human CA isoenzymes are written in capital roman letters and numbers, whereas their genes are written in italic letters and arabic numbers. Alternatively, “MN” is used herein to refer either to carbonic anhydrase isoenzyme IX (CA IX) proteins/polypeptides, or carbonic anhydrase isoenzyme 9 (CA9) gene, nucleic acids, cDNA, mRNA etc. as indicated by the context.
The MN protein has also been identified with the G250 antigen. Uemura et al. [35] states: “Sequence analysis and database searching revealed that G250 antigen is identical to MN, a human tumor-associated antigen identified in cervical carcinoma (Pastorek et al., 1994).”
Zavada et al., International Publication Number WO 93/18152 (published Sep. 16, 1993) and U.S. Pat. No. 5,387,676 (issued Feb. 7, 1995), describe the discovery of the MN gene and protein. The MN gene was found to be present in the chromosomal DNA of all vertebrates tested, and its expression to be strongly correlated with tumorigenicity. In general, oncogenesis may be signified by the abnormal expression of CA IX protein. For example, oncogenesis may be signified: (1) when CA IX protein is present in a tissue which normally does not express CA IX protein to any significant degree; (2) when CA IX protein is absent from a tissue that normally expresses it; (3) when CA9 gene expression is at a significantly increased level, or at a significantly reduced level from that normally expressed in a tissue; or (4) when CA IX protein is expressed in an abnormal location within a cell.
The MN protein is now considered to be the first tumor-associated carbonic anhydrase isoenzyme that has been described. The carbonic anhydrase family (CA) includes eleven catalytically active zinc metalloenzymes involved in the reversible hydration-dehydration of carbon dioxide: CO2+H20HCO3−+H+. CAs are widely distributed in different living organisms. The CAs participate in a variety of physiological and biological processes and show remarkable diversity in tissue distribution, subcellular localization, and biological functions [1, 2, 27]. Carbonic anhydrase IX, CA IX, is one of the most recently identified isoenzymes [3,4]. Because of the CA IX overexpression in transformed cell lines and in several human malignancies, it has been recognized as a tumor-associated antigen and linked to the development of human cancers [5-7].
CA IX is a glycosylated transmembrane CA isoform with a unique N-terminal proteoglycan-like extension [4]. Through transfection studies it has been demonstrated that CA IX can induce the transformation of 3T3 cells [4]. Recent studies have revealed that CA IX not only participates in cell adhesion, but also can be induced in hypoxia via the HIF-1 protein binding to the hypoxia-responsive element of the MN promoter [8, 9]. The transcription of the MN gene is negatively regulated by the von Hippel-Lindau tumor suppressor gene in renal cell carcinoma cells [28]. The protein product of the von Hippel-Lindau tumor suppressor gene interacts with the ubiquitin ligase complex that is responsible for targeting HIF-1α for oxygen-dependent proteolysis [29, 30]. Thus, low levels of oxygen lead to stabilization of HIF-1α, which in turn leads to the increased expression of MN [9]. Areas of high expression of MN in cancers are linked to tumor hypoxia as reported in many cancers and incubation of tumor cells under hypoxic conditions leads to the induction of MN expression [9-14].
Many studies have confirmed the diagnostic/prognostic utility of MN, using the MN-specific monoclonal antibody (MAb) M75 in diagnosing/prognosing precancerous and cancerous cervical lesions [6, 37, 38, 39, 55]. Immuno-histochemical studies with the M75 MAb of cervical carcinomas and a PCR-based (RT-PCR) survey of renal cell carcinomas have identified MN expression as closely associated with those cancers and indicates that MN has utility as a tumor biomarker [6, 36, 38]. In various cancers (notably uterine cervical, ovarian, endometrial, renal, bladder, breast, colorectal, lung, esophageal, head and neck and prostate cancers, among others), CA IX expression is increased and has been correlated with the microvessel density and the levels of hypoxia in some tumors [10, 11].
In tissues that normally do not express MN protein, CA IX positivity is considered to be diagnostic for preneoplastic/neoplastic diseases, such as, lung, breast and cervical cancers [12-14]. However, among those cancerous tissues, higher MN expression often indicates a better prognosis. Previous studies have reported that there is an inverse correlation between CA IX expression and stage and grade in some tumors, including clear cell RCC [40], cervical carcinoma [39], colorectal tumors [7], and esophageal cancer [52]. Of these studies, the three that were non-RCC-related found that low expression of CA IX correlated with poor prognostic factors, such as lymph node metastases and depth of invasion. Bretheau et al. 1995 [41] reported the poor prognosis of RCC patients with high grade and stage tumors, which according to Uemura et al. [40] would be expected to express CA IX at lower levels. Bui et al. [42; International Publication No. WO 03/089659] reported that “low” CA IX (≦85%) staining was an independent poor prognostic factor for survival for patients with metastatic RCC.
Very few normal tissues have been found to express MN protein to any significant degree; those MN-expressing normal tissues include the human gastric mucosa and gallbladder epithelium, and some other normal tissues of the alimentary tract [45, 15, 16]. Immunohistochemical analysis of the normal large intestine revealed moderate CA IX staining in the proximal colon, with the reaction becoming weaker distally. The staining was confined to the basolateral surfaces of the cryptal epithelial cells, the area of greatest proliferative capacity. As CA IX is much more abundant in the proliferating cryptal epithelium than in the upper part of the mucosa, it may play a role in control of the proliferation and differentiation of intestinal epithelial cells. Cell proliferation increases abnormally in premalignant and malignant lesions of the colorectal epithelium, and therefore, is considered an indicator of colorectal tumor progression. [43, 44]. Interestingly, CA9 deficient mice develop gastric hyperplasia which is associated with increased proliferation [17], raising the question, whether the putative pathophysiological role of CA IX in gastric cancer development and progression is different from the one observed in cancers of non-gastric origin.
Gastric cancer is the second most common cause of cancer-related deaths worldwide [22, 23]. Despite its decreasing incidence it remains a great challenge for clinicians and oncologists. In recent years various groups have analysed the genetic and molecular changes leading to gastric cancer. Those changes include, among others, the overexpression of oncogenes, such as growth factor receptors K-sam and c-met, the loss of certain tumor suppressor genes, such as APC and p53, as well as alteration of adhesion molecules, including E-cadherin and the catenins [22-26]. Recently, the group of carbonic anhydrases and especially CA IX have received increasing attention [2]. However, studies to date investigating CA IX expression in gastric mucosa, which normally overexpresses CA IX, have provided only diagnostic analysis, associating the presence of gastric cancer with diminished levels of CA IX or loss of CA IX.
Disclosed herein is a surprising finding that has led to novel and inventive prognostic methods for gastric cancer and related cancers, that are diagnosed by the loss or reduction of the CA IX expression, that is abundant in corresponding normal tissue. Surprisingly, among such cancers, it was found that CA IX expression that is higher than the absent or significantly reduced levels of CA IX expression considered diagnostic for gastric and related cancers, indicates a poorer prognosis for patients that have been diagnosed with such cancers, particularly when such higher CA IX expression is found at the invasion fronts of such cancers. Disclosed herein is the surprising finding that in gastric cancer and related cancers, a higher expression of CA IX indicates a poorer prognosis for afflicted patients, particularly when expressed at the invasion front of the cancer.